Motor neurons (MN) in patients with amyotrophic lateral sclerosis (ALS) degenerate through unclear mechanisms. Apoptosis could be a mechanism for this neuronal loss. Previous work on MN death mechanisms in ALS has lacked cellular resolution for MN-specific events. We propose to study the levels of selected cell death molecules in MN from humans with ALS and transgenic ALS mice using laser capture microdissection and proteomic analyses with ProteinChip arrays. The profiles of human and mouse ALS MN will be compared to MN induced to degenerate (by sciatic nerve avulsion) through a process that is unequivocally apoptosis and is p53- and Bax-dependent and also involves mitochondrial accumulation, oxidative stress, DNA damage, and caspase-3 activation. We hypothesize that MN degeneration in human and mouse ALS is structurally a nonclassical form of apoptosis that is DNA damage-induced and mediated by p53, or its homologue p73, and caspases. The upstream mechanisms for MN death that we will study in human and mouse ALS and in axotomy will be the formation of DNA lesions (abasic sites and single- and double-strand breaks) and signaling pathways leading to accumulation of p53/p73. We will measure, in human and mouse ALS, different types of damage to chromosomal and mitochondrial DNA. We will use an innovative method (the comet assay) to quantify different forms of DNA lesions in single MN and to determine if known and potentially new pharmacotherapies (creatine and selenomethionine) for mouse ALS attenuate the formation of DNA lesions. Our preliminary data also implicate DNA damage-responsive kinases in the mechanisms of MN death in human and mouse ALS and in axotomy. We will measure the activation of selected DNA damage-responsive protein kinases (ATM and c- Abl) in MN in human and mouse ALS and in avulsion. We will use mouse ALS and avulsion models of MN degeneration to directly identify upstream mechanisms leading to MN death. We will test the hypothesis that MN degeneration is stimulated by accumulation of DNA single-strand breaks and mediated by ATM and/or c- Abl activation and p53/p73 activation. We will determine if avulsion-induced MN apoptosis is dependent on ATM. Pharmacological inhibition of c-Abl with STI571 and inhibition of p53/p73 with pifithrin-V will be used to modify the degeneration of MN in ALS mice. We will also measure DNA repair enzymes in human and mouse ALS MN because failed or defective DNA repair in MN could lead to the accumulation of DNA lesions and cellular degeneration. This work is essential for the further understanding of the biological substrates and molecular mechanisms of MN death and the pathogenesis of ALS and is critical for the identification of novel molecular targets and new drug therapies for the treatment of ALS.